The yeast two-hybrid system has proven to be a powerful method for the discovery of specific protein interactions in vivo. See generally, Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, N.Y., 1997. The yeast two-hybrid technique is based on the fact that the DNA-binding domain and the transcriptional activation domain of a transcriptional activator contained in different fusion proteins can still activate gene transcription when they are brought into proximity to each other. In a yeast two-hybrid system, two fusion proteins are expressed in a yeast cell. One has a DNA-binding domain of a transcriptional activator fused to a test protein. The other, on the other hand, includes a transcriptional activating domain of the transcriptional activator fused to another test protein. If the two test proteins interact with each other in vivo, the two domains of the transcriptional activator are brought together reconstituting the transcriptional activator and activating a reporter gene controlled by the transcriptional activator. See FIG. 1. See also, e.g., U.S. Pat. No. 5,283,173.
Because of its simplicity, efficiency and reliability, the yeast two-hybrid system has gained tremendous popularity in many areas of research and development. Yeast cells are eukaryotic cells. The interactions between mammalian proteins detected in the yeast two-hybrid system typically are bona fide interactions that occur in mammalian cells under physiological conditions. As a matter of fact, numerous mammalian protein-protein interactions have been identified using the yeast two-hybrid system. The identification of such protein-protein interactions has contributed significantly to the understanding of many biological processes.
In addition, the yeast two-hybrid system can also be used in drug screening to identify compounds capable of modulating a particular protein-protein interaction. Indeed, a modified version of the yeast two-hybrid system, the so called “reverse two-hybrid assay,” is one of the most powerful assays for identifying chemical inhibitors of protein-protein interactions. In essence, a reverse yeast two-hybrid assay employs one or more counterselectable reporters such that yeast cells harboring such reporters can be positively selected only when a target protein-protein interaction is disrupted by a test compound. Various reverse two-hybrid systems are disclosed in, e.g., U.S. Pat. Nos. 5,525,490; 5,733,726; 5,885,779; and 5,965,368.
However, these reverse two-hybrid methods have serious drawbacks. For example, the counter-selectable signals caused by a test compound typically are reversible. Consequently, continued dissociation of target proteins is often required during an assay in order to provide an adequate selection signal. However, continued dissociation of target proteins during an assay is typically difficult, if not impossible, to achieve. The test compounds that dissociate target proteins may not be stable and can decompose during the assay. Prolonged exposure of test compounds to host yeast cells may lead to activation of the host cellular machineries to metabolize or eject the compounds. Some test compounds may only be effective at certain phases of host cell growth and become ineffective as the host cell grows. Another factor that exacerbates the problems is that the effective concentration of a test compound is typically unknown a priori and can be within a rather narrow range. In light of these constraints, a range of concentrations must be examined under conditions where the concentrations are stable. Such exhaustive testing is antithetical to high-throughput screening. Instead, a single concentration is often selected for testing in liquid assays, notwithstanding the possibility that the effective concentration may be significantly different from the tested concentration. Alternatively, solid agar assays can be used, in which a concentrated solution of a test compound is spotted onto a lawn of cells and diffusion establishes a dynamic concentration gradient. Still, given the instability of the concentration gradient, compounds that are active over a narrow concentration range may escape identification.
Therefore, there is a great need for two-hybrid systems that can be used in large-scale high throughput screening assays without the above shortcomings.